Up-to-date materials engineering demands production of high purity metals having homogeneous particle-size distribution in the range of from 50 to 500 nm. Homogeneous size and form, as well as spherical shape are preferred for microelectronic and pharmaceutical technologies. Among these, elemental selenium plays a significant role. Elemental selenium has more known allotropic modifications: red colored amorphous and crystalline selenium, as well as metallic grey selenium. The electric conductivity of grey selenium increases up to thousand fold by illumination. Consequently, it is extensively used, for example, as material of photo sensor detectors and photocopiers.
Furthermore, selenium is an essential micronutrient for animals and humans, but in higher dose it can be toxic. In humans, selenium is a trace element which functions as cofactor for reduction of antioxidant enzymes such as glutathione peroxidases and certain forms of thioredoxin reductase found in animals and some plants. Bioavailability and toxicity are strongly related to the chemical form of selenium (Hartikainen, H. 2005: Biogeochemistry of selenium and its impact on food chain quality and human health. J. Trace Elem. Med. Biology 18: 309-318.). Inorganic selenium(IV) is less toxic than selenium(VI) in the soil plant system as it was described by Széles (Széles, É. 2007: Selenium-Speciation In Soil And Plant Samples Doctoral thesis University of Debrecen). Organic selenium forms are less toxic but highly bioavailable forms, therefore they have significant importance in functional foods, food and feed supplements. Some plant, like cabbage and onion can convert the inorganic selenium to organic, but others can not. Yeast is applied for the industrial production of selenium-methionine from inorganic selenium, but we have limited information about the conversion of inorganic selenium by bacteria (Rayman, M. P. 2004: The use of high-selenium yeast to raise selenium status: how does it measure up? Br J Nutr., 92: 557-573).
Selenium is a metalloid element that is chemically similar to sulfur and tellurium and in nature exists in four oxidation states, −2, 0, +4, and +6. The last two states occur in aqueous media as the soluble oxyanions selenite [SeO32− or Se(IV)] and selenate [SeO42− or Se(VI)]. Selenium also has unusual photo-optical and semiconducting physical properties and has industrial applications in devices such as photocopiers and microelectronic circuits. Recent interest in the field of nanotechnology has stimulated research into the chemical synthesis of selenium nanowires that are composed of elemental selenium [Se(0)] (Abdelouas, A., W. L. Gong, W. Lutze, J. A. Shelnutt, R. Franco, and I. Moura. 2000. Using cytochrome cc3 to make selenium nanowires. Chem. Mater. 12:1510-1512; Gates, B., B. Mayers, B. Cattle, and Y. Xia. 2002. Synthesis and characterization of uniform nanowires of trigonal selenium. Adv. Funct. Mater. 12:219-227; Gates, B., B. Mayers, B. Grossman, and Y. Xia. 2002. A sonochemical approach to the synthesis of crystalline selenium nanowires in solutions and on solid supports. Adv. Mater. 14:1749-1752).
Disadvantageously, chemically manufactured metal particles have irregular shape, generally the produced crystals are surrounded by edges, and the particles' size varies in a wide range. The capability of selenium-respiring bacteria to produce elemental selenium has been disclosed in the art but only in strictly anaerobic conditions, and in microorganisms isolated from geological and environmental samples.
To date, more than 15 diverse species of Bacteria and Archaea have been described that grow anaerobically by linking the oxidation of organic substrates or H2 to the dissimilatory reduction of selenium oxyanions (Oremland, R. S., and J. F. Stolz. 2000. Dissimilatory reduction of selenate and arsenate in nature, p. 199-224. In D. R. Lovley (ed.), Environmental metal-microbe interaction. ASM Press, Washington, D.C., Stolz, J. F., and R. S. Oremland. 1999. Bacterial respiration of selenium and arsenic. FEMS Microbiol. Rev. 23:615-627). The end products of these reactions are the red, amorphous or monoclinic allotropes of Se(0), which accumulate in spent medium because the microorganisms reduce the 10 to 20 mM selenate or selenite provided to Se(0). Respiratory reductases for Se oxyanions contain molybdenum and are associated with the plasma membrane (18).
Reports on the reduction of selenite to elemental selenium by sulphate reducing, selenium-respiring bacteria has been published first time in the 1990s (Tomei, F. A., Barton, L. L., Lemanski, C. L., Zocco, T. G., Fink, N. H., Sillerud, L. O., 1995. Transformation of selenate and selenite to elemental selenium by Desulfovibrio desulfuricans. J. Ind. Microbiol. 14, 329-336.). The possibility of industrial application of the analyzed bacteria has not been investigated, and its industrial adaptability has not been considered. In 1999 J. Kessi et al. (J. Kessi, m. Ramuz, E. Wehrli, M. Spycher, and R. Bachofen (1999) Reduction of Selenite and Detoxification of Elemental Selenium by the Phototrophic Bacterium Rhodospirillum rubrum Applied and Environmental Microbiology, November 1999, Vol. 65, No. 11. p. 4734-4740) used anaerobic phototrophic purple non-sulphur bacterium called Rhodospirillum rubrum in their experiments, which can be found in still waters and swamps in nature, and which is also able to bind elemental nitrogen of the atmosphere. This bacterium utilizes CO2, H2 and NH3 in anaerobic conditions while using sulphate as source of sulphur. Medium used in experiments contained 40-120 mg/L selenium in the form of sodium selenite. The reduction process' duration was 60-80 hours. In the process orange-red colored amorphous selenium was produced. Disadvantageously, the size of the so formed selenium spheres varied in wide range depending on conditions, ranging from 50 to 200 nm in a single specimen.
In 2003, R. S. Oremland et al. examined the capability of three anaerobic, selenium-respiring bacteria (Sulfurospirillum barnesii, Bacillus selenitireducens and Selenihalanaerobacter shrifti) to produce elemental selenium (Ronald S. Oremland, Mitchell J. Herbel, Jodi Switzer Blum, Sean Langley, Terry J. Beveridge, Pulickel M. Ajayan, Thomas Sutto, Amanda V Ellis, Seamus Curran (2003) Structural and Spectral Features of Selenium Nanospheres Produced by Se-Respiring Bacteria Applied and Environmental Microbiology, Vol. 70, No. 1 Jan. 2004, p. 52-60). Bacteria analyzed were anaerobic organisms living in watery environment. It has been found that the species analyzed produced red colored selenium forms characterized by molecular formulae S8 and S6. The drawback of the process is that it can only be used for producing the red form, and as yet there is no established practice and experience for the industrial application of the employed organisms.
In 2004, G. Sarret et al. evaluated the selenium production of selenium-respiring soil bacterium Ralstonia metallidurans (Geraldine Sarret, Laure Avoscan, Marie Carriere, Richard Collins, Nicolas Geoffroy, Francine Carrot, Jacques Coves, Barbara Gouget (2004) Chemical Forms of Selenium in the Metal-Resistant Bacterium Ralstonia metallidurans CH34 Exposed to Selenite and Selenate Applied and Environmental Microbiology, Vol. 71, No. 5 May 2005, p. 2331-2337). They found that the time required for production of elemental selenium in medium comprising selenium in the form of sodium selenite was 100-120 hours, which is a significantly long duration. Klonowska et al. have reported the selenite producing capability of the selenium-respiring bacterium Shewanella in 2005 (Klonowska, A., Heulin, T., Vermeglio, A., 2005. Selenite and tellurite reduction by Shewanella oneidensis. Appl. Environ. Microbiol. 71, 5607-5609.). Shewanella, unlike many other microorganisms, does not grow on oxygen and nutrients, but catabolizes metals and excretes free electrons as by-products. This bacterium can live in soil and water as well, and is able to select proper energy source among diverse materials. There are drawbacks of employing this bacterium, namely the absence of an established practice of its industrial application, furthermore, its proliferation is much slower than that of other strains used in industrial fermentation processes.
In a report published in 2007 by Lee et al. investigated effects of temperature and dissolved oxygen on selenium-producing mechanism of the selenium-respiring Shewanella sp. strain HN-41 (Ji-Hoon Lee, Jaehong Han, Heechul Choi, Hor-Gil Hur (2007). Effects of temperature and dissolved oxygen on Se(IV) removal and Se(0) precipitation by Shewanella sp. HN-41 Chemosphere, doi:10.1016/j.chemosphere. 2007.02.062 in press). The red selenium spheres produced by the bacterium were sized 150-200 nm, and its size was not controllable by oxygen supply.
Summarizing, it can be stated that in each work performed in the art, the ability of anaerobic, selenium-respiring bacteria originating from soil, water or geological samples to produce elemental selenium was investigated. Investigators didn't consider or suggest this microbiological approach as potentially resulting in a suitable industrial scale method for producing elemental selenium nanospheres. Selenium nanosphere producing strains described in the art are all selenium-respiring bacteria producing elemental selenium in anaerobic conditions as a result of their anaerobic respiratory activity, none of which bacteria is used for industrial production; introduction of such bacteria into industrial scale production would raise numerous problems as manipulations with pathogenic and/or toxic microorganisms require specific conditions and licenses.
In each so far investigated case, the microbiologically produced selenium was red selenium microbial production of the grey modification of selenium is not known in the art. In each case, production of elemental selenium required 80-200 hours. Medium generally contained 100-200 mg/L selenium in the form of sodium selenite.